ORIGINAL RESEARCH published: 12 May 2021 doi: 10.3389/fvets.2021.678968

Is Encephalitozoon cuniculi of Significance in Young With Neurological Signs?

Tamar S. de Boer, Montse M. Diaz Espineira and Paul J. J. Mandigers*

Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands

Encephalitozoon cuniculi is a microsporidium belonging to the phylum Microspora. A few reports have described the clinical significance of E. cuniculi infection in young dogs. In American and Japanese household populations, the seroprevalence was found to be 21%, indicating its wide-spread existence. To evaluate the clinical significance of E. cuniculi in a cohort of young dogs with neurological signs, the presence of the organism and possible response to treatment were studied. Over a 1-year period, all young dogs (< 3 years old) that were referred with neurological signs were examined for the presence of E. cuniculi. Dogs were selected if revealed a clearly elevated IgM titer (>100) and/or if an EDTA-blood sample and/or urine sample tested positive

Edited by: by polymerase chain reaction (PCR). Sixteen dogs with various neurological signs were John Henry Rossmeisl, included in this study. Additional work-up included magnetic resonance imaging and Virginia Tech, United States cerebrospinal fluid analysis, but these revealed no abnormalities or indication of infection. Reviewed by: All dogs were treated with fenbendazole for 10–30 days. Neurological signs disappeared Kari Foss, University of Illinois at completely in five dogs, 11 dogs continued to show neurological signs, and five dogs Urbana-Champaign, United States deteriorated and were euthanized, after which necropsy was performed in three. At Iva Langrova, Czech University of Life Sciences necropsy no evidence of an E. cuniculi infection was found. We concluded that, although Prague, Czechia IgM titers and PCR indicated an E. cuniculi infection, it is most likely of limited clinical *Correspondence: significance in young dogs. Paul J. J. Mandigers [email protected] Keywords: cerebrospinal fluid analysis, dog, Encephalitozoon cuniculi, fenbendazole, infection, treatment 2

Specialty section: This article was submitted to INTRODUCTION Veterinary Neurology and Neurosurgery, Encephalitozoon cuniculi is a microsporidium belonging to the phylum Microspora (1, 2). a section of the journal E. cuniculi is an obligate intracellular parasite that can infect , including humans, in Frontiers in Veterinary Science which it is considered an (2–4), but it is most frequently seen in rabbits Received: 10 March 2021 (4). Mammals can get infected by ingestion of contaminated water or food, inhalation of spores or Accepted: 16 April 2021 through in utero (1, 5). The spores infect enterocytes and spread through blood or the Published: 12 May 2021 lymphatic system. Predilection organs are among others lung, liver, and brain (1, 5). It takes Citation: 3–5 weeks for the life cycle to complete. There are three known strains of E. cuniculi. Strain I was Boer TSd, Diaz Espineira MM and isolated from rabbits, strain II from rodents, and strain III from dogs (6). Mandigers PJJ (2021) Is Infected rabbits do not always show clinical signs, as a latent infection is possible. If clinical Encephalitozoon cuniculi of Significance in Young Dogs With signs are seen in rabbits, these signs are subdivided into neurological signs, renal issues, ophthalmic Neurological Signs? problems, or combinations of these. The neurological signs are characterized by vestibular disease Front. Vet. Sci. 8:678968. (torticollis, ataxia, rolling, and paresis). The renal form is characterized by chronic renal failure doi: 10.3389/fvets.2021.678968 and its sequalae, such as weight loss, polyuria, polydipsia, and/or urinary incontinence. The

Frontiers in Veterinary Science | www.frontiersin.org 1 May 2021 | Volume 8 | Article 678968 Boer et al. Significance of Encephalitozoon cuniculi in Dogs? ophthalmologic form is characterized by intraocular lesions, electromyographic studies (EMG), and/or specific DNA testing, including , uveitis, and hypopyon (5). if indicated. If any abnormality was found that was indicative It was first observed that this agent could affect dogs in for a specific treatment (for instance a dog with an atlanto- 1952, when Plowright (7) reported a granuloma containing axial instability or a hydrocephalus), the dog was not eligible for the parasite in the brains of two puppies (7). Although the this study. number of reports of E. cuniculi in dogs is small, these reports concern young or immunocompromised dogs. The clinical signs Inclusion and Exclusion Criteria observed are the same as those reported in rabbits and are renal Dogs were only included if the routine biochemistry was free of (4), ophthalmologic (8), and/or neurological in nature (9). The abnormalities, if toxoplasmosis and neospora titers were found to neurological signs described are blindness, ataxia, and seizures be negative, and if additional work-up (CSF, MRI, DNA testing, (9, 10). etc.) did not exclude an infection as a possible cause of the Previous studies have demonstrated infections with the neurological signs. Hence, if the MRI and/or CSF analysis was parasite in dogs. In both an American (11) and a Japanese indicative of an inflammatory disorder, the dog was eligible for population (12) of dogs, the seroprevalence for E. cuniculi was this study. 21%. It has been suggested that adult dogs do not develop Dogs were only included and defined as possibly infected with clinical disease after infection with E. cuniculi, because they are E. cuniculi if serology showed a clearly elevated IgM titer and/or believed to be immunocompetent (13), but this does not apply to if the PCR of the EDTA sample and/or urine sample was found young dogs. to be positive. The IgM was considered elevated if the value was Although E. cuniculi has been identified in a number of studies 100 or more, while a titer of <30 was regarded as negative. as a possible cause of neurological signs in young dogs, it is not routinely tested in the first line and referral clinic. However, Treatment and Response to Treatment this is based on the earlier studies, remarkedly as it may be a Included dogs were treated with fenbendazole only, at a dose of differential diagnosis in young dogs. This study evaluated the 50 mg/kg bodyweight, for a period of 10 days, after which the clinical significance of E. cuniculi in a cohort of young dogs clinical outcome was evaluated (4, 14). If a dog did not respond to with neurological signs, by investigating its presence and possible treatment, the dog was considered to be a non-responder. At this response to a fenbendazole treatment. stage, the owner was offered a second treatment, or an additional treatment based on the presumed diagnosis. For instance, an MATERIALS AND METHODS anti-epileptic if the dog presented epilepsy, glucocorticosteroids if an immune-mediated process was suspected, etc. If an owner Dogs elected a second or third treatment, the dog’s clinical outcome Over a period of 1 year, all young dogs (<3 years of age) that were was again evaluated after 10 days. If a dog deteriorated regardless referred to the Department of Clinical Sciences at the Faculty of of the additional treatment and the owner elected euthanasia, a Veterinary Medicine, Utrecht University, The Netherlands, were post-mortem examination was requested. examined for the presence of E. cuniculi. Dogs were considered eligible for inclusion if they showed neurological signs, such as Analytic Method epilepsy, generalized ataxia, tremors, or dyskinesias in which an The three strains of E. cuniculi were grown on Vero or MRC infection would be a logical differential diagnosis. All dogs were cells using RPMI 1640 medium under 5% CO2. After cultivation, evaluated by a single investigator (PJJM). the spores were harvested, collected, concentrated, washed, and brought to a concentration of 109, after which they were Procedure suitable for use in IFAT and a soluble ELISA antigen tests After obtaining informed consent from each owner, a normal (Elisa-kit product-nr LA1003-AB01 from the EVL Laboratory; diagnostic work-up was performed. In all dogs, a complete www.evlonline.org). The ELISA antigen was titrated on Greiner hematology and biochemistry panel (University Veterinary of Costar strip plates after production using a checkerboard Diagnostic Laboratory; www.uvdl.nl) as well as serology titration, with known high (∼1,350) and low titer (∼450) sera for toxoplasmosis and neospora (Veterinary Microbiology and anti-rabbit horseradish peroxidase (from the EVL laboratory; Diagnostic Centre; www.uu.nl/vmdc) was performed. E. cuniculi www.evlonline.org; Cat: Do RA IgG-HRPO RTU19-05). After was evaluated by obtaining additional serum, EDTA-blood, determining the optimal coating dilution, ELISA plates were and urine samples; these samples were examined for the coated and blocked, stabilized, and stored. A mixed coating was presence of relevant (IgM and IgG) and of parasite used for standard ELISA research. As a substrate, a 2-component antigen by means of an immunofluorescence test substrate RTU from the EVL laboratory (www.evlonline.org) was (IFAT) and polymerase chain reaction (PCR), respectively used (Cat: TMBA-2019-06 and TMBB-2019-06 from the EVL (see section Treatment and Response to Treatment), at Laboratory). The reaction was allowed to proceed for 10 min ◦ a commercial veterinary laboratory (European Veterinary at 21 C; then, the reaction was stopped by adding 50 µl of Laboratory [EVL]; www.evlonline.org). diluted H2SO4, and the absorbance of the colored solution was Based on the neurological signs and neuro-localization, all read at 450 nm. dogs underwent additional diagnostic steps, such as magnetic To assess the results, a sample ratio (positive control vs. resonance imaging (MRI), cerebrospinal fluid (CSF) analysis, standard method) or cutoff titer (TG-ROC plot) and 95%

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TABLE 1 | Included dogs, age in months at the time of referral, and the duration of neurological signs present.

Case number Breed Age Illness Neurological signs (months) duration (months)

1 Labrador 32 1 Head tremor, vestibular signs 2 Labradoodle 28 2 Generalized tremors and ataxia 3 Dutch Shepherd 4 1 Head tremor, epileptic seizures 4 French Bulldog 8 1 Head tremor, epileptic seizures, generalized ataxia 5 Cesky Fousek 39 1 Head tremor, epileptic seizures, generalized ataxia 6 German Shepherd 4 1 Tonic-clonic seizures 7 33 7 Paroxysmal dyskinesia 8 Pug 19 10 Head tremor, vestibular signs 9 3 1 Generalized ataxia, vestibular signs 10 Old English Bulldog 23 4 Head tremor, epileptic seizures 11 Nova Scotia Duck Tolling Retriever 3 6 Tremor front legs, ataxia of hindlegs 12 Jack Russel Terrier 4 1 Head tremor, epileptic seizures 13 Norfolk Terrier 7 1 Abnormal behavior, epileptic seizures 14 Tervueren Shepherd 8 1 Ataxia of hindlegs 15 Border Collie 8 2 Generalized ataxia, vestibular signs 16 Rottweiler 24 15 Ataxia of hindlegs

confidence interval was used. Batch validation was performed Neurological Signs with pools of wild-type infected, type-selected, lyophilized sera Neurological signs in 14 of the 16 dogs were suggestive of at (1020). To test field samples, two HRPO-conjugates of IgM and least an intracranial neuro-localization (Table 1). Twelve of the IgG and different controls were used. IgM determinations were 16 dogs had multiple varied neurological signs, including head only made after pre-absorption of IgG. Titrations (100, 300, 900, tremor (seven dogs), epileptic seizures (focal or generalized, in 2700) of the obtained samples were performed with IgM, starting seven dogs), generalized tremors (two dogs), vestibular signs at 1:50. (four dogs), and generalized ataxia (five dogs). Only four dogs The PCR was based on an internal transcribed spacer and/or showed a single neurological sign: one with only tonic-clonic tyrosine phosphatase sequences (made by the EVL seizures, one with paroxysmal dyskinesia, and two with ataxia of Laboratory and classified; www.evlonline.org). Conventional the hindlegs (Table 1). PCR was performed for type identification, while real-time PCR with ultra-black probes was also performed. Lyophilized Laboratory Findings infected cell cultures were used as controls at two levels of two Routine hematology and biochemistry revealed some different types of PCRs (standard methods, from Jena Bioscience, abnormality in all dogs. Fourteen dogs had a positive PCR Jena, Germany). in their EDTA-blood sample, of which one also a PCR-positive Statistical Analysis urine sample (no other dog had a positive urine sample) (Table 2). Of these 14 dogs, four dogs had an elevated IgM titer Results were statistically analyzed with IBM SPSS Software, of 100, all others had IgM titers below 30. Two of the 14 dogs version 24 (IBM, Chicago, IL, USA). Descriptive statistics had an IgG titer of 300, and seven a titer of 100; the remaining and a Pearson chi-squared test were performed. Results were five had titers below 30. Two dogs had an elevated IgM titer (300 considered significant when the p-value was < 0.05. and 1,000), but a negative PCR for both urine and EDTA-blood sample. The IgG titer in these two dogs was 300. Both dogs RESULTS showed signs of generalized ataxia, head tremor, and epileptic Table 2 Dogs seizures ( ). Forty-two young dogs (<3 years of age) were eligible for this study, but only 16 met the final inclusion criteria. The dog breeds Additional Diagnostic Evaluation varied, with no breed overrepresented. The age of onset had a In nine of the 16 dogs, an MRI scan and CSF analysis were median of 8 months (range 3–39 months). The duration of illness performed. None of the MRI scans revealed any abnormality, and had a median of 1 month (range 1–15 months) (Table 1). CSF was regarded to be normal in all nine dogs (Table 2).

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TABLE 2 | Laboratory and additional findings and outcomes.

Case number IgM titer IgG titer PCR EDTA MRI CSF protein Treatment period Outcome Pathology

1 <30 <30 Positive - 10 days Improved NA, alive 2 100 100 Positive No abn. 0.3 20 days Improved NA, alive 3 <30 100 Positive - 10 days Improved NA, alive 4 1,000 300 Negative No abn. 0.35 10 days Improved NA, alive 5 300 300 Negative - 30 days Improved NA, alive 6 100 100 Positive No abn. 0.2 10 days Slight NA, alive improvement 7 30 100 Positive No abn. 0.2 10 days No improvement NA, alive 8 <30 100 Positive No abn. 0.28 20 days No improvement NA, alive 9 <30 30 Positive No abn. 0.18 10 days No improvement NA, alive 10 30 100 Positive - 10 days No improvement NA, alive 11 30 300 Positive No abn. 0.3 10 days No improvement NA, alive 12 100 100 Positive - 20 days No improvement, Not allowed died 13 <30 <30 Positive - 30 days No improvement, Not allowed died 14 100 300 Positive No abn. 0.33 20 days No improvement, Yes, no died abnormalities 15 30 30 Positive - 10 days No improvement, Yes, no died abnormalities 16 <30 <30 Positive No abn. 0.4 10 days No improvement, Yes, no died abnormalities

Dogs included with their IgM, IgG titers, PCR results in the EDTA blood sample, as well as the MRI findings and CSF protein amount. The CSF cell count was not abnormal in all cases. Case number 14 also had a positive urine PCR. Additionally, the treatment period, clinical outcome, and if necropsy was performed, the outcome are also listed.

Response to Treatment DISCUSSION All dogs were treated according to the protocol. Five dogs responded to the treatment, of which two responded within Although some reports suggest that E. cuniculi is of significance 10 days, two within 20 days, and one during the third period in young dogs, it is not routinely tested. This is remarkable, as of fenbendazole treatment. All five dogs had signs suggestive previous studies not only described the clinical signs in young of a multifocal or generalized neurological disorder. Two of dogs (8–10), but also a rather high seroprevalence of 21% in these responders had a negative PCR, but a clearly elevated IgM American (11) and Japanese (12) populations, suggesting that titer. Two had a positive PCR and elevated IgM and/or IgG, dogs do come into contact with the parasite. For this reason, while one dog only had a positive PCR, but without IgM and we evaluated the clinical significance of this parasite in a group IgG elevation. of young dogs. In rabbits with clinical signs, a diagnosis is Eleven dogs did not improve, regardless of treatment. All considered confirmed if E. cuniculi spores are detected in the these dogs received a second course of fenbendazole, but as urine or if the parasite is found to be present in the blood or urine they did not respond, an additional treatment, based on the sample based on PCR (15). Whether this also applies to dogs is presumed diagnosis, was started. Only one out of these 11 not known. dogs did respond after the additional treatment with an anti- The 16 examined dogs were all found to be positive either epileptic. The condition of five of these 10 non-responding by PCR (14 dogs) or by clearly elevated IgM titer (two dogs). dogs deteriorated, and euthanasia was elected. Three of these The latter is suggestive of an immune response to the parasite, dogs were necropsied, but in none of these cases was the while the first demonstrates that the parasite was ingested. histopathology abnormal, and there was no indication of an E. As most of our dogs had either an elevated IgM titer (6 of cuniculi infestation. 16) or IgG titer (11 of 16), we concluded that there was an In all dogs, serology and PCR were repeated after either 10 (the immune response to the parasite in these dogs. The clinical five responding dogs) or after 20 days (the 11 non-responding signs observed in the dogs were in accordance with previous dogs), and in all dogs the PCR was found to have become studies (9, 10) and were in all dogs, except for four, suggestive negative. There was no statistically significant difference for dogs of multifocal neuro-localization. In young dogs, the differential that had improved compared to those that did not improve in diagnosis would be intoxication, inflammatory, either infectious terms of MRI findings (p = 0.39), CSF protein level (p = 0.43), or or immune-mediated, generalized or multi-focal neoplasia, treatment outcome and period (p = 0.82). and/or degenerative disorders. Based on the protocol followed,

Frontiers in Veterinary Science | www.frontiersin.org 4 May 2021 | Volume 8 | Article 678968 Boer et al. Significance of Encephalitozoon cuniculi in Dogs? the most likely differential diagnosis in these dogs would be an be treated with fenbendazole or (4, 14). However, inflammatory etiology. the uptake of fenbendazole is less than that of albendazole, Interestingly, the MRI scans and CSF analysis were normal which may underlie the lack of response observed. However, in nine of the 16 dogs. There are no detailed reports describing albendazole is embryotoxic, teratogenic, and can cause serious MRI findings and CSF analysis in dogs with E. cuniculi infection. side effects in dogs, and hence, we chose fenbendazole as it is However, one report in a human case states that both CSF and registered as safe to be used in dogs (19). MRI findings were clearly abnormal (16). Furthermore, the CSF analysis in rabbits diagnosed with E. cuniculi infection is also CONCLUSION clearly abnormal with elevated cell counts as well as protein levels (17); this was not the case with the dogs in this study. Despite the The parasite E. cuniculi was found to be present in 16 negative CSF analysis, it would have been of interest to perform a dogs presenting various neurological signs. However, MRI and PCR on the CSF for the various neurotropic infections. However, CSF analysis were not found to be abnormal in nine dogs, at the time of this study this test was not available. three dogs were necropsy-negative, and there was a lack of Interestingly five dogs improved after the fenbendazole response to treatment in 11 dogs. Based on the current results, treatment. Four of these dogs suffered from head tremor, which E. cuniculi is most likely of limited clinical significance in is also classified as an idiopathic head tremor (18), and is known young dogs. to resolve by itself in many cases. Nevertheless, all four these dogs also had other neurological signs, suggestive of a multifocal DATA AVAILABILITY STATEMENT localization, and therefore the head tremor was less likely to be idiopathic. Eleven dogs did not respond to treatment, and The raw data supporting the conclusions of this article will be an additional treatment had to be initiated. Despite this, five made available by the authors, without undue reservation. dogs showed deterioration of neurological signs and the owners elected euthanasia. Three of these dogs were necropsied but ETHICS STATEMENT no abnormalities suggestive of an E. cuniculi infection were observed. In rabbits, the diagnosis is confirmed if granulomatous Ethical review and approval was not required for the animal study inflammation with spores is found in renal, hepatic, and/or brain because all dogs were referred cases with neurological deficits. tissue at post-mortem (5), as also described by Plowright (7) and Written informed consent was obtained from the owners for the Snowden et al. (10). participation of their animals in this study. This study posed the question of whether E. cuniculi is a true in dogs. All dogs included into this study were AUTHOR CONTRIBUTIONS infected with E. cuniculi, but MRI and CSF analysis revealed no abnormalities in nine of these dogs nor in the three necropsied TB performed this study as her master thesis. MD co-supervised dogs. And there was a lack of response to treatment in 11 dogs. and reviewed with PM the study. PM set up the study, examined Based on these results, E. cuniculi is most likely of limited clinical all dogs, supervised, and reviewed the analysis. All authors read significance in young dogs. and approved the final manuscript. This study has limitations. First of all, the prevalence of E. cuniculi in dogs and rabbits in the Netherlands is not studied and ACKNOWLEDGMENTS therefore unknown. E. cuniculi as a disease in rabbits is frequently observed in the Netherlands but exact morbidity and mortality We wish to thank Mr. Rob van Herwijnen of European figures are sadly unknown. Secondly the study population is Veterinary Laboratory (Woerden, The Netherlands) for small. It may be of interested to perform this study together providing us with technical specification of the analytical with a prevalence study in other countries as well. And if E. methods, Miriam Duijvestijn (DVM) of the Veterinary cuniculi was the cause of neurological signs in these examined Diagnostic Microbiology Centre of the Faculty of Veterinary dogs, the lack of response could be because of our choice of Medicine, Utrecht University, for her critical input, and Editage fenbendazole treatment. Rabbits suffering from E. cuniculi can for English language editing.

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